1. Volume 4, Issue 3, Pages 445-458 (March 2015)
Enhanced Hematopoietic Stem Cell Function Mediates Immune Regeneration following Sex Steroid Blockade 
Danika M. Khong, Jarrod A. Dudakov, Maree V. Hammett, Marc I. Jurblum, Sacha M.L. Khong, Gabrielle L. Goldberg, Tomoo Ueno, Lisa Spyroglou, Lauren F. Young, Marcel R.M. van den Brink, Richard L. Boyd, Ann P. Chidgey 
Stem Cell Reports 
Volume 4, Issue 3, Pages (March 2015)
DOI: /j.stemcr
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2. Stem Cell Reports 2015 4, 445-458DOI: (10.1016/j.stemcr.2015.01.018)
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3. Figure 1
SSA Increases the Number of Multilineage HSCs in Middle-Aged Mice
(A–D) Lin−SCA1+cKIT+ (LSK) BM can be subdivided into populations of LT-HSCs (CD34−FLT3−), ST-HSCs (CD34+FLT3−), and MPPs (CD34+FLT3+). The MPP population can be further fractionated based on FLT3 and CD62L expression for analysis of LMPPs (FLT3hiCD62L+). Absolute number of LT-HSCs (A), ST-HSCs (B), MPPs (C), and LMPPs (D) (n = 5–12/group/time point).
(E) Concatenated flow cytometry plots, gated on Lineage− cells, and absolute number of FLT3− LSK cells, 1 year after surgical SSA of 9-month male mice (n = 5/group).
(F) LSK cells were FACS purified from untreated CD month; CD month mice 7 days following surgical shSSA (d7shSSA); or CD month mice 7 days following surgical SSA (d7SSA) (n = 6 recipients/group/dose) and graded doses of cells were transferred into lethally irradiated congenic CD45.1 recipients along with 5 × 105 CD45.1+ supporting BM cells. Multilineage reconstitution (>1% B cell, T cell, macrophage, and granulocyte) was analyzed 12 weeks after transplant and the frequency of repopulating cells was calculated by Poisson statistics.
Bar graphs represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figures S1 and S2.
Stem Cell Reports 2015 4, DOI: ( /j.stemcr )
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4. Figure 2
SSA Enhances the Functional Repopulation Potential of HSCs Derived from Middle-Aged Mice
(A–C) CD45.1 BM cells were harvested and pooled from 9-month d7shSSA, (n = 5 recipients/donor group) or 9-month d7SSA (n = 5 recipients/donor group) and 2.5 × 106 cells were transferred with an equal dose of untreated 2-month CD45.2 BM cells into lethally irradiated 2-month CD45.2 recipients. Reconstitution was analyzed at the designated time points. (A) Concatenated donor (CD45.1) versus competitor (CD45.2) flow cytometry profiles of spleen 17 weeks after transplant. (B) Total peripheral reconstitution measured by CD45.1 in spleen or peripheral blood over 120 days. (C) Lineage-specific reconstitution of B220+ B cells, TCRβ+ T cells, Gr1+CD11b+ granulocytes, and Gr1loCD11b+ monocyte/macrophages in the spleen at 28 and 120 days after transplant.
(D–G) 2,000 FACS purified CD45.1 LSK cells from untreated 2-month; 9-month d7shSSA; or 9-month d7SSA were transferred along with 2 × 106 BM cells from untreated 2-month CD45.2 competitors into lethally irradiated CD45.2 recipients (n = 5–7/donor group). (D) Total number of CD45.1 donor-derived cells in the BM of recipients 28 days after transplant. (E) Proportion of CD45.1+ whole BM, LSK, CLP, and MCP cells in the BM 28 days after transplant. (F) Concatenated donor (CD45.1) versus competitor (CD45.2) flow cytometry profiles of spleen 19 weeks after transplant. (G) Peripheral reconstitution in 28, 70, and 135 days after transplant. ∗Compared with 9-month shSS A mice. ˆCompared with untreated 2-month mice.
(H) Lineage repopulation was measured among B220+ B cells, TCRβ+ T cells, Gr1+CD11b+ granulocytes, and Gr1loCD11b+ monocyte/macrophages.
Results are expressed as mean ± SEM. ∗/ˆp < 0.05, ∗∗/ˆˆp < 0.01, ∗∗∗/ˆˆˆp < See also Figure S2.
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5. Figure 3
SSA Enhances the Self-Renewal Capacity of HSCs Derived from Middle-Aged Mice
2,000 CD45.1 purified LSK cells from CD month untreated, 9-month d7shSSA, or 9-month d7SSA were transferred along with 2.5 × 106 untreated 2-month CD45.2 BM cells into lethally irradiated 2-month CD45.2 primary recipients (n = 6 recipients/donor group, one individual recipient per individual donor). 12 weeks after transplant, primary recipient BM was harvested, and 2.5 × 106 BM cells were transplanted along with an equal dose of untreated 2-month CD45.2 BM cells into lethally irradiated 2-month CD45.2 secondary recipients (n = 12 recipient/donor groups, two recipients per individual donor). Reconstitution of secondary recipients was measured 12 weeks after transplant.
(A) Proportion of CD45.1+ donor cells in BM 12 weeks following transplant in primary and secondary recipients (∗Compared with 9-month d7shSSA mice within primary or secondary recipients. ˆCompared to untreated 2-month mice within either primary or secondary recipients. †Compared to 9-month d7shSSA between primary and secondary recipients).
(B) Proportion of donor-derived CD150+CD34−FLT3− LT-HSCs and CD34+FLT3hiCD62L+ LMPPs in the BM of secondary recipients 12 weeks after transplant.
(C) Absolute number of donor-derived LT-HSCs and LMPPs in the BM of secondary recipients 12 weeks after transplant.
(D) Concatenated flow cytometric profiles of total donor reconstitution, as well as donor B220+ B cells, TCRβ+ T cells, Gr-1+CD11b+ granulocytes, and Gr1loCD11b+ in the spleen of secondary recipients 12 weeks after transplant.
(E) Absolute number of CD45.1+ donor cells in the spleen of secondary recipients 12 weeks after transplant.
(F) 12 weeks after secondary transplant, 2.5 × 106 BM cells from secondary recipients was transplanted into tertiary recipients at a 1:1 ratio with competitor CD45.2+ BM, and peripheral reconstitution was assessed at 12 weeks. Concatenated FACS plots of the proportion, and the absolute number of CD45.1+ donor cells, in tertiary recipients (n = 6/group).
Results are expressed as mean ± SEM. ∗/ˆp < 0.05, ∗∗/ˆˆp < 0.01.
Stem Cell Reports 2015 4, DOI: ( /j.stemcr )
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6. Figure 4
SSA Promotes Lymphoid Differentiation Potential of HSCs Derived from Middle-Aged Mice
(A and B) 5 × 106 CD45.1 BM cells from untreated 2-month; 9-month d7shSSA; or 9-month d7SSA were transferred into unablated 2-month Il7ra−/− recipients (n = 6 recipient/donor groups, one recipient per donor). (A) Concatenated flow cytometry plots displaying total donor reconstitution as well as donor Gr1+CD11b+ granulocyte, Gr1loCD11b+ monocyte/macrophage, B220+ B cells, and TCRβ+ T cells reconstitution at day 42. (B) Absolute number of donor cells as well as the number of donor B cells and T cells at day 42.
(C and D, and G and H) 2,000 CD45.1 LT-HSCs from untreated 2-month; 9-month d7shSSA; or 9-month d7SSA were transferred into unablated 2-month Il7ra−/− recipients (n = 6 recipient/donor groups, one recipient per donor) and donor-derived hematopoiesis measured in the spleen 70 days after transfer. (C) Concatenated flow cytometry profiles showing total donor reconstitution as well as donor Gr1+CD11b+ granulocyte, Gr1loCD11b+ monocyte/macrophage, B220+ B cells, and TCRβ+ T cells reconstitution. (D) Absolute number of donor-derived cells in the periphery of Il7ra−/− recipients.
(E and F) 2,000 LMPPs from untreated CD month; CD month d7shSSA); or CD month d7SSA were transferred into unablated 2-month Il7ra−/− recipients (n = 6 recipient/donor groups) and donor-derived hematopoiesis measured in the spleen 70 days after transfer. (E) Concatenated flow cytometry profiles showing total donor reconstitution as well as donor B220+ B cells, and TCRβ+ T cells reconstitution. (F) Absolute number of donor-derived cells in the periphery of Il7ra−/− recipients.
(G) Concatenated flow cytometry plots displaying donor reconstitution and absolute numbers of donor Gr1+CD11b+ granulocytes.
(H) Concatenated flow cytometry plots displaying donor reconstitution and absolute numbers of donor Gr1loCD11b+ monocyte/macrophages.
Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S3.
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7. Figure 5 Intrinsic Molecular Changes within HSCs after SSA
(A) 10,000 LT-HSCs from 9-month shSSA or 9-month SSA mice 2 or 7 days after surgery were plated onto OP9-DL1 cells and T cell differentiation assessed at day 12. Concatenated FACS plots gated on CD4−CD8−CD3−.
(B–D) LT-HSCs from 9-month d2shSSA or 9-month d2SSA mice were FACS purified, and transcriptome analysis was performed (n = 3 biological replicates, where each replicate included cells pooled from 10–20 animals). (B) Volcano plot outlining gene expression between 9-month d2shSSA and 9-month d2SSA LT-HSCs. 251 genes were significantly altered (p < 0.05; >1.5-fold change), 55 upregulated and 196 downregulated. (C and D) Significant changes between 9-month d2shSSA and 9-month d2SSA LT-HSCs in genes involved with cell-cycle regulation (C) or differentiation (D).
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8. Figure 6
SSA Induces Molecular Changes to the Niche and Its Ability to Support Hematopoiesis
Transcriptome analysis was performed on purified sinusoidal CD45−TER119− cells from 2- and 9-month untreated mice, 9-month shSSA control, and 9-month SSA mice at days 2, 4, 7, and 10 after surgery (n = 1, ten mice pooled/sample).
(A) Pattern discovery (correlation >0.7) identified 132 probes that demonstrated an increasing gene expression trend toward young profiles following castration of middle-aged mice.
(B) qPCR of Foxo1 expression in pre-OBLs purified from untreated 2- or 9-month mice, and 9-month d2shSSA or 9-month d2SSA mice.
(C–E) Expression of Cxcl12, Kitlg, Spp1, Ang1, Jag1, Vcam1, Bmp4, Il7, Tgfb, and Igf1 in endothelial cells (C), pre-OBLs (D), and OBLs (E) from 9-month d2SSA and 9-month d7SSA. Expression is represented as 9-month d7SSA relative to 9-month d7shSSA control mice, n = 3–11 independent experiments.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figure S4.
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9. Figure 7
SSA Promotes the Ability of the Niche to Support Hematopoiesis
Lethally irradiated 9-month d7shSSA or 9-month d7SSA were reconstituted with either 5 × 105 or 5 × 106 BM cells from untreated 2-month CD45.1 donors (n = 5–6/group).
(A) Concatenated flow cytometry plots showing the proportion of donor LSK cells at 28 days after transplant.
(B) Absolute number of donor-derived LSK cells in the BM 28 days after transplant.
(C) Concatenated flow cytometry plots showing total donor reconstitution in the spleen at days 28 and 84 after transplant.
(D) Absolute number of donor-derived cells in the spleen at days 28 and 84 after transplant.
(E) Absolute number of donor-derived Gr1+CD11b+ granulocyte, Gr1loCD11b+ monocyte/macrophage, B220+ B cells, and TCRβ+ T cells in the spleen 84 days after transplant.
Results are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.
Stem Cell Reports 2015 4, DOI: ( /j.stemcr )
Copyright © 2015 The Authors Terms and Conditions